CAJ | 학술논문

目的利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1与UL128-642 bp编码蛋白相互作用.结论成功构建pGBKT7 UL128-519 bp和pGBKT7 UL128-642 bp,并应用酵母双杂交系统分别筛选出EFEMP2和THY-1与UL128-519 bp和UL128-642 bp编码蛋白相互作用,在所筛选得到的相互作用蛋白之间未发现有相同的蛋白,UL128-519 bp和UL128-642 bp所编码的蛋白在HCMV感染致病过程中可能发挥不同的作用.
목적 이용효모쌍잡교계통종인태뇌cDNA문고중사선여량충불동전록결구적인거세포병독(HCMV)UL128편마단백상호작용적단백,비교량자상호작용단백지간적이동점.방법 통과3'RACE화5'RACE기술확증출량충HCMV UL128편단,기대소분별위519 bp화642 bp,병장기성공구건도효모유이표체재체pGBKT7중.장이상량충효모표체재체분별전화도효모균AH109중,재장문고DNA전화도이함유효모표체재체적AH109중,사선여량충편단대소불동적UL128편마단백상호작용적인태뇌단백,병대사선득도적양성극륭진행측서화생물신식학분석.결과 사출EFEMP2여UL128-519 bp편마단백상호작용,THY-1여UL128-642 bp편마단백상호작용.결론 성공구건pGBKT7 UL128-519 bp화pGBKT7 UL128-642 bp,병응용효모쌍잡교계통분별사선출EFEMP2화THY-1여UL128-519 bp화UL128-642 bp편마단백상호작용,재소사선득도적상호작용단백지간미발현유상동적단백,UL128-519 bp화UL128-642 bp소편마적단백재HCMV감염치병과정중가능발휘불동적작용.
Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.